The Notch signaling pathway regulates a diverse array of cell functions (Kopan et al., Cell 137, 216-233 (2009)). Four Notch receptors have been identified in mammals, i.e., Notch 1-4, that share basic structural elements that include an extracellular domain, a transmembrane domain, and an intracellular domain. Similarly, the canonical ligands of Notch share certain structural similarities but a number of non-canonical ligands of Notch have also been identified (Kopan et al., Cell 137, 216-233 (2009)). The five canonical ligands in mammals are Delta-like 1, Delta-like 3, Delta-like 4, Jagged1 and Jagged2. Binding of a Notch ligand to the extracellular domain of a Notch receptor sets a signaling cascade in motion that begins with proteolytic cleavage at the extracellular S2 site by an alpha secretase of the ADAM (a disintegrin and metalloprotease) family. Cleavage at S2 is followed by proteolytic cleavage by a gamma secretase at the intracellular S3 site, which results in release of the intracellular domain and downstream events that ultimately activate Notch-dependent transcription factors such as Hes1 and Hey.
Because aberrant Notch expression and signaling has been implicated in a number of diseases, including cancer (Koch et al., Cell. Mol. Life Sci. 64, 2746-2762 (2007)), modulators of Notch signaling have been investigated as possible therapeutic agents for such diseases. For example, gamma secretase inhibitors have been tested in clinical trials for their effectiveness in treating various malignancies (Shih et al, Cancer Res. 67, 1879-1882 (2007)). Gamma secretase inhibitors prevent cleavage at S3 and thereby prevent signaling through Notch receptors. However, gamma secretase inhibitors do not distinguish individual Notch family members and therefore inhibit signaling through multiple receptors at once, as well as through unrelated pathways (Beel et al., Cell. Mol. Life Sci. 65, 1311-1334 (2008)). Moreover, administration of gamma secretase inhibitors is associated with intestinal toxicity marked by weight loss and intestinal goblet cell metaplasia, indicative of a role for Notch in determining cell fate by maintaining proliferation of intestinal crypt progenitor cells and prohibiting differentiation to a secretory cell fate (see van Es et al., Nature 435:959-963 (2005)). Similarly, inhibition of both Notch1 and Notch2 signaling via conditional Notch gene knockout (Riccio et al., EMBO Rep. 9:377-383 (2008)) or via antagonist antibody inhibition (US Patent Application Publication No. 2010/0080808) also causes intestinal goblet cell metaplasia.
The mouse intestinal epithelium provides an important model for studying tissue regeneration. Continuous turnover of the epithelium is supported by intestinal stem cells (ISCs) located near the base of the crypts. Genetic lineage tracing studies have led to the identification of distinct ISC populations, including crypt base columnar cells (CBCs) that are marked by Lgr5 expression, a Wnt target gene (Barker, van Es et al. 2007). CBCs reside at the bottom of crypts, occupying cell positions +1 through +5 from the base, where they are intercalated between post-mitotic Paneth cells, which constitute the stem cell niche (Sato, van Es et al. 2011). CBCs contribute to all intestinal cell types, including the secretory and absorptive lineages, through a population of rapidly proliferating intermediates known as transit-amplifying (TA) cells (Barker, van Es et al. 2007). Continuous replacement and sloughing of old cells leads the intestinal epithelium to renew approximately every 5 days.
Development of the small intestine and adult intestinal homeostasis requires canonical Wnt signaling. Lef/Tcf4, the transcription factor that mediates canonical Wnt signaling, is essential for the formation of proliferative compartments in prospective crypt regions of neonatal mice (Korinek, Barker et al. 1998). Lef/Tcf4 is also required for adult intestinal homeostasis (van Es, Haegebarth et al. 2012), as is the Wnt effector β-catenin (Fevr, Robine et al. 2007). Conversely, administration of the Wnt signaling agonist R-spondin1 leads to expansion of the ISC compartment (Yan, Chia et al. 2012), which can mitigate loss of ISCs during chemoradiation (Zhou, Geng et al. 2013). In adult animals, the central role of Wnt signaling is highlighted by the Wnt-dependent expression of numerous ISC markers, including Lgr5. (de Lau, Barker et al. 2011). In addition to its role in maintaining ISCs, Wnt signaling confers competence for the secretory fate decision. Wnt signaling plays specific roles in Paneth cell differentiation (Andreu, Colnot et al. 2005; van Es, Jay et al. 2005; Andreu, Peignon et al. 2008; van Es, Haegebarth et al. 2012), whereas overexpression of the Wnt inhibitor DKK1 leads to loss of all secretory cells (Pinto, Gregorieff et al. 2003).
The Notch pathway affects intestinal homeostasis by regulating CBCs and by promoting the absorptive cell fate. Compromising Notch signaling in adult mice with the γ-secretase inhibitor DAPT, which blocks the conversion of the Notch receptor into its transcriptionally active state, causes a loss of proliferating Lgr5-positive CBCs and an overall increase in secretory cells (VanDussen, Carulli et al. 2012). Secretory cell hyperplasia in the gut also occurs with deletion of the Notch effector Rbp-j (van Es, van Gijn et al. 2005; VanDussen, Carulli et al. 2012). Conversely, the activation of constitutive Notch signaling in the small intestine of perinatal mice causes an expansion of the proliferative compartment as well as a decrease in the number of secretory cells (Fre, Huyghe et al. 2005; Stanger, Datar et al. 2005). Genetic evidence suggests that Notch signaling exerts its negative regulatory effect on secretory cell differentiation entirely through repression of Math1/Atoh1, a transcription factor required for the formation of secretory cells (Yang, Bermingham et al. 2001), as conditional deletion of Math1 rescues the Rbp-j loss of function phenotype (Kim and Shivdasani 2011). However, while Math1 is up-regulated in the absence of Notch (VanDussen, Carulli et al. 2012), the signal(s) required for positively maintaining normal levels of Math1 in the small intestine are unknown.
There is a need in the art for anti-Notch pathway therapeutic regimens that lack the toxicity associated with inhibition of Notch receptors.